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T Yoshimura

Publications and source records attributed to T Yoshimura.

At least 397 records · Page 22Linked to original sources
PubMed1994

Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification.

The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme. These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.

Aspartate Aminotransferases↗
PubMed1994

Interferon-gamma induces different subunit organizations and functional diversity of proteasomes.

To obtain information on the role of proteasomes in the immune system, we examined the effect of a major immunomodulatory cytokine, gamma interferon (IFN-gamma), on the expressions, structures, and functions of proteasomes. IFN-gamma greatly increased the levels of the mRNAs encoding LMP2 and LMP7, putative immuno-proteasome subunits encoded by genes within the class II MHC region, and these two subunits synthesized were assembled completely into the proteasomal multi-subunit complex in various types of human cells. The subunit organization of proteasome changed in response to IFN-gamma stimulation, due to assembly of newly synthesized subunits through up- and down-expressions of at least 6 proteasome genes including LMP2/LMP7 without change in the structure of pre-existing proteasomes. Interestingly, IFN-gamma dramatically stimulated the trypsin-like and chymotrypsin-like activities of the multifunctional proteasome and depressed the peptidylglutamyl-peptide-hydrolyzing activity, without affecting the activity for ATP-, ubiquitin-dependent proteolysis. These results indicate that IFN-gamma modifies not only the structural organization of the proteasome, but also its functions. Based on these findings, we discuss the role in the antigen processing/presentation pathway of proteasomes with functional diversity acquired through alteration of their subunit assembly in response to IFN-gamma stimulation.

Adenosine Triphosphate↗
PubMed1994

Fusion of phospholipid vesicles induced by clathrin: modulation of fused liposome size.

The effects of lipid composition, temperature, and ionic strength on critical events in the membrane fusion reaction induced by clathrin, such as membrane binding and self-association of clathrin, membrane aggregation, and actual fusion, and the size of fused liposomes, were examined using large unilamellar vesicles (LUV) containing acidic phospholipids. When membrane fusion and aggregation of LUV with different lipid compositions were initiated at 25 degrees C in 0.1 M NaCl by lowering the pH of the medium from 7.40 to 4.75 in the presence of clathrin, two types of reaction processes were observed: fusion and aggregation of LUV containing phosphatidylserine and phosphatidic acid occurred slowly with a long fusion lag-phase and reached a high level, whereas the two reactions of LUV containing phosphatidylglycerol and phosphatidylinositol were induced faster with a much shorter fusion lag-time and leveled off in a shorter time. Similar differences in the fusion reactions were observed in media at different temperatures and ionic strengths with any type of LUV: slow and extensive aggregation and fusion occurred at low temperature and/or high ionic strength, but faster, less extensive reactions occurred at high temperature and/or low ionic strength, indicating that the two types of reaction pattern are due to the dependencies of the aggregation and fusion reactions on the temperature and ionic strength, but not on the lipid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Clathrin↗
PubMed1994

Construction and characterization of chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase.

Phenylalanine dehydrogenase of Thermoactinomyces intermedius acts preferentially on L-phenylalanine and L-tyrosine, whereas leucine dehydrogenase of Bacillus stearothermophilus acts almost exclusively on L-leucine and some other branched-chain L-amino acids. The two enzymes share a sequence similarity (47%). Aiming at elucidation of the mechanism of substrate recognition by the two amino acid dehydrogenases, we have genetically constructed a chimeric enzyme consisting of an N-terminal domain of phenylalanine dehydrogenase containing the substrate-binding region and a C-terminal domain of leucine dehydrogenase containing the NAD(+)-binding region. The chimeric enzyme purified to homogeneity acted on phenylalanine with a specific activity of 6% of that of the parental phenylalanine dehydrogenase and showed a broad substrate specificity in the oxidative deamination, like phenylalanine dehydrogenase. However, it acted much more effectively than phenylalanine dehydrogenase on isoleucine and valine. Its Km values for L-phenylalanine and L-leucine were similar to those of phenylalanine dehydrogenase. The substrate specificity of the chimeric enzyme in the reductive amination was an admixture of those of the two parent enzymes. These results suggest that the two domains of phenylalanine dehydrogenase and leucine dehydrogenase probably can fold independently. Accordingly, their chimera forms a new active enzyme which consists of their N- and C-terminal domains containing the substrate- and coenzyme-binding regions, respectively. However, the two domains of chimeric enzyme interact and communicate with each other to form a new active site and consistently show the new substrate specificity.

Amino Acid Oxidoreductases↗
PubMed1994

Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.

A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1994

Immunohistochemical study of heparan sulfate proteoglycan in adenocarcinomas of the pancreas.

The prognosis for carcinoma of the pancreas is extremely poor. One of the characteristics of this tumor is its invasion of the surrounding tissues. Reduction of glycoprotein is considered to be conducive to invasion of the basement membrane by carcinoma cells. Heparan sulfate proteoglycan (HSPG), a kind of glycoprotein, is an important component of basement membrane. In this study, the relation between HSPG and carcinoma of the pancreas was examined by using the immunohistochemical method, and the survival rate of pancreatic adenocarcinoma was evaluated. We found that some carcinomas contained little or no HSPG. The poorer the differentiation of an adenocarcinoma of the pancreas, the lower was its content of HSPG. The level of HSPG was significantly different in carcinomatous and in noncarcinomatous cells. There was a close correlation among the content of HSPG, the degree of differentiation of carcinomas of the pancreas, and the survival time. HSPG seems to be useful in prognosis of adenocarcinoma of the pancreas.

Adenocarcinoma↗
PubMed1994

K-ras and p53 alterations in genomic DNA and transcripts of human pancreatic adenocarcinoma cell lines.

We analyzed 15 human pancreatic adenocarcinoma cell lines for alterations of the K-ras and the p53 genes and their transcripts. In 11 cell lines (73.3%), point mutations of the K-ras gene were found at codon 12 in exon 1. In 9 cell lines one allele was mutated and the other was wild type, and both the alleles were expressed into mRNA. In one cell line both alleles of codon 12 were mutated to TGT and GTT, respectively, but only TGT was transcribed into mRNA. Alterations in mRNA of the p53 gene were detected in 10 cell lines (66.7%). Analysis of the genomic sequence of the p53 gene revealed that the alterations consisted of 6 cases of base pair substitutions and 1 case of 1-bp deletion in evolutionarily conserved exons 5 to 8, 2 cases of splicing mutations in exon 4, and 1 case of novel deletion from exons 2 to 9. In 14 cell lines (93.3%), alterations were identified in the K-ras or p53 gene. Of these, 4 cell lines harbored K-ras mutations without p53 alteration, whereas 3 cell lines exhibited p53 alterations without K-ras mutation. Thus, it is suggested that activation of the K-ras gene and inactivation of the p53 gene are strongly and cooperatively associated with pancreatic carcinogenesis.

Adenocarcinoma↗
PubMed1994

Increased expression of the monocyte chemoattractant protein-1 in bronchial tissue from asthmatic subjects.

The expression of the monocyte chemoattractant protein (MCP-1), a member of the chemokine family of low molecular weight cytokines, was assessed by immunohistochemistry in bronchial biopsies from 12 asthmatic and 12 normal subjects. Both a monoclonal antibody (F9) and a polyclonal antibody were employed to detect MCP-1, while the mouse myeloma protein (MOPC21) was used as a negative control. Strong positive reactions for MCP-1 were seen in the bronchial epithelium. Subepithelial macrophages, blood vessels, and bronchial smooth muscle were also stained. Hue-saturation-intensity color image analysis was used to quantify reactions of the monoclonal antibody in the epithelial and subepithelial layers. With the monoclonal antibody, asthmatic biopsies showed 51.8 +/- 3.7% (mean +/- SEM) of the epithelium staining positively, whereas normal subjects reacted much less, with 6.4 +/- 1.9% of the epithelium staining (P < 0.0001); there was no overlap between the two groups. Likewise, staining was increased in the subepithelium of asthmatic airway biopsies, with 11.5 +/- 3.1% and 2.0 +/- 1.0% staining positively in asthmatic and normal subepithelium, respectively, (P < 0.002). There was a significant correlation between staining of the epithelium and subepithelium (r = 0.77, P < 0.001). The polyclonal anti-MCP-1 antibody also gave strong reactions in the epithelium and subepithelium, with 34.0 +/- 7.8% of the asthmatic and 1.6 +/- 1.0% of the normal bronchial epithelium staining positively (P < 0.0001). These increased levels of MCP-1 in the asthmatic airways suggest that they may play a role in macrophage recruitment and activation and thereby contribute to the inflammatory pathology of bronchial asthma.

Adult↗
PubMed1994

Role of interleukin-1 in endotoxin-induced lung injury in the rat.

The effects of the recombinant interleukin-1 receptor antagonist (rIL-1ra) on the systemic vascular and lung injury following intraperitoneal Salmonella enteritidis lipopolysaccharide (LPS) were determined in male Sprague-Dawley rats. Initial experiments identified that maximal mortality occurred with an intraperitoneal LPS dose of 20 mg/kg, and this dose was used in subsequent experiments. Albumin permeability, measured in an ex vivo perfused heart-lung preparation from the rats 2 h after injection of LPS, was increased with endotoxin as was the wet:dry weight ratio. Pretreatment of the rats with intravenous rIL-1ra, 1 to 10 mg/kg, followed by a continuous intravenous infusion at 30 to 50 micrograms/kg/min resulted in restoration of blood pressure at 100 min following endotoxin administration. Moreover, coadministration of rIL-1ra with endotoxin totally prevented the rise in albumin permeability of the pulmonary vasculature and the increase in wet:dry lung weight ratios observed in rats treated with LPS alone. LPS injected intraperitoneally caused a marked decrease in circulating leukocyte count, an effect not reversed by rIL-1ra. RNA extraction of whole-lung homogenates revealed that mRNA for IL-1 beta was constitutively expressed in the absence of endotoxin, but transcripts increased progressively from 0.5 to 2 h after endotoxin administration. Increases in mRNAs for tumor necrosis factor-alpha (TNF-alpha) and for macrophage inflammatory protein-2 (MIP-2), a potent neutrophil chemoattractant, were also observed from 0.5 until 2 h after endotoxin administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Differences in circadian photosensitivity between retinally degenerate CBA/J mice (rd/rd) and normal CBA/N mice (+/+).

Using the magnitude of phase shift of circadian locomotor rhythms induced by a single pulse of white fluorescent light, we compared the sensitivity of the circadian system to light in retinally degenerate mice and in normal mice. In the first experiment, phase response curves (PRCs) for 10-lux white light were generated in CBA/J mice with retinal degeneration (rd/rd) and CBA/N mice with normal retinas (+/+). Although large phase delays early in the subjective night and small phase advances in the late subjective night were observed in CBA/N mice, CBA/J mice showed only small phase delays early in the subjective night. In the second experiment, we found that the magnitude of phase shifts at circadian time (CT) 16 for delays and CT 24 for advances in CBA/J mice became larger with increasing light intensity, and that CBA/J mice could show the same amount of phase shift as CBA/N mice when higher intensities were used. These findings indicate that the differences in the shapes of PRCs are not due to differences in the nature of the oscillating system, but to differences in circadian photosensitivity between these strains. Because the genetic background for the rd loci was not completely identical in the CBA/N and CBA/J mice, it was possible that genes other than the rd gene might have caused different photosensitivity in these mice. Therefore, in the last experiment, we studied the circadian photosensitivity in F1 hybrids between CBA/N and CBA/J mice and in the backcross progeny with different genotypes (+/rd and rd/rd) obtained from the crossing between F1 hybrids between CBA/N and CBA/J mice and in the backcross progeny with different genotypes (+/rd and rd/rd) obtained from the crossing between F1 and CBA/J mice. In these mice with heterogeneous genetic backgrounds as well, mice with retinal degeneration were always less sensitive to light, suggesting that reduced circadian photosensitivity is caused by retinal degeneration. These results are discussed in relation to recent findings in retinally degenerate C57BL mice, which have been found to have normal circadian sensitivity to light.

Animals↗
PubMed1994

Inhibition of carbon tetrachloride-induced hepatotoxicity by neopterins.

We investigated the inhibitory effects of neopterin (NP) and its reduced form, 5,6,7,8-tetrahydroneopterin (NPH4), on carbon tetrachloride (CCl4)-induced hepatotoxicity. In in vivo experiments, intraperitoneal administration of NP or NPH4 significantly inhibited the elevation of plasma alanine aminotransferase activity induced by CCl4 in mice. In in vitro experiments using cultured rat hepatocytes, CCl4 induced in a manner which was both time- and dose-dependent lactate dehydrogenase release, and the addition of NP or NPH4 to the culture-medium significantly inhibited its release from cells. NPH4, but not NP, reacted directly with a stable radical, 1,1-diphenyl-2-picrylhydrazyl. These results suggest that NP and NPH4 inhibit CCl4-induced hepatotoxicity through different mechanisms.

Alanine Transaminase↗
PubMed1994

Lymphocyte stimulation test with tetrazolium-based colorimetric assay for diagnosis of drug-induced allergic hepatitis.

The lymphocyte stimulation test (LST) is useful for diagnosing drug-induced allergy and identifying the causative drug. In this study, we examined the usefulness of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) as a marker for LST in diagnosing drug allergy. In a basic study using normal peripheral blood mononuclear cells, the normal range of stimulation index (SI) was 0.92-1.38, and the mean SI for all drugs tested was 1.134 +/- 0.111 (mean +/- S.D.). The cut-off value of SI for diagnosis of drug allergy was thus set at over mean + 2S.D. for possibly positive, and at over mean + 3S.D. as a definitely positive reaction. Forty-six cases of suspected drug-induced allergic hepatitis involving 85 drugs were diagnosed by this assay, and the possibly positive and definitely positive rates were 54.3% (SI > or = 1.4) and 41.3% (SI > or = 1.5), respectively. A clinical study was made of 113 patients with diagnosed drug-induced allergic hepatitis. Forty-nine (43%) of the patients were male and 64 (57%) were female. In 85% of cases the allergic reaction occurred within one month of taking medication, but there were a number of cases in whom onset occurred after long-term incubation. The main clinical symptoms were jaundice, itching, eruption, fever, and general malaise. In about 75% of cases glutamic oxaloacetic transaminase (GOT) or glutamic pyruvic transaminase (GPT) returned to normal range within one month after medication was halted.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Plasma concentration of atrial natriuretic peptide and brain natriuretic peptide during normal human pregnancy and the postpartum period.

Increases in blood volume are observed during normal gestation and these are reversed shortly after delivery. Although both atrial (A-type) natriuretic peptide (ANP) and brain (B-type) natriuretic peptide (BNP) have been described, the role of these peptides in pregnancy and the postpartum period are unclear. This study was designed to examine the effects of pregnancy, labour and delivery on plasma levels of ANP and BNP. Plasma levels of ANP and BNP were determined during normal pregnancy, 30 min after separation of the placenta (immediately postpartum) and between 5 and 72 h postpartum (late postpartum; puerperium). Since the assay sensitivity was 20 pg/ml plasma (for both ANP and BNP), values less than this were assigned a value of 20 pg/ml to calculate means. Plasma levels of ANP and BNP were significantly higher at term pregnancy than during the first trimester (ANP increased from 20 +/- 0.2 to 57 +/- 10 pg/ml (S.E.M.), P < 0.001; BNP increased from 25 +/- 2 to 49 +/- 9 pg/ml, P < 0.01). The plasma level of ANP then rose to 157 +/- 38 pg/ml 30 min after separation of the placenta, being significantly (P < 0.01) higher than that seen at term pregnancy. It declined significantly (P < 0.001) to 32 +/- 3 pg/ml in the late postpartum period. In contrast, the plasma level of BNP 30 min after separation of the placenta was 80 +/- 25 pg/ml, and increased to 116 +/- 17 pg/ml in the late postpartum period, significantly (P < 0.01) higher than the level at term pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Locked-in syndrome and abnormal orientation of the right vertebral artery in a young man.

A 35-year-old man developed a cerebral infarction and experienced transient ischemic attacks originating from the vertebrobasilar artery, as well as locked-in syndrome. He recovered with minimal neurological deficit. On cerebral angiography, the orientation of the right vertebral artery was markedly abnormal as it entered the foramina of the transverse process at the level of the third cervical vertebra. We concluded that the abnormal orientation of the vertebral artery caused the thrombosis and that the transient ischemic attacks, locked-in syndrome and cerebral infarction were brought about by thromboemboli originating in this artery.

Adult↗
PubMed1994

Generalized normolipemic plane xanthomatosis associated with relapsing polychondritis.

Generalized normolipemic plane xanthomatosis is a rare cutaneous disorder, frequently associated with reticuloendothelial diseases and some disorders with inflammation. Relapsing polychondritis is also a rare disease that shows an association with various immune-mediated diseases. We report a case of generalized normolipemic plane xanthomatosis associated with relapsing polychondritis in a 56-year-old Japanese man. We have already reported the clinical picture of relapsing polychondritis as well as an increase in urinary glycosaminoglycans excretion in this patient. During subsequent treatment with various immunosuppressive therapy, including prednisone, methotrexate, azathioprine, or aurothiomalate, multiple elevated yellowish erythematous plaques appeared on his neck approximately 32 months after the onset of relapsing polychondritis. Histologically, these eruptions consisted of perivascular neutrophilic infiltrate with nuclear dust and multiple foam cells among collagen bundles, compatible with those of generalized normolipemic plane xanthomatosis. This combination of two rare diseases has not been reported in the literature to our knowledge.

Humans↗
PubMed1994

Expression and localization of messenger RNA and protein for monocyte chemoattractant protein-1 in human malignant glioma.

Expression of monocyte chemoattractant protein-1 (MCP-1) in human glioma cell lines and surgical specimens was studied by Northern blot analysis, reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. The samples tested consisted of 11 human glioma cell lines and eight specimens of human malignant glioma (seven from glioblastomas and one from a malignant ependymoma). Messenger ribonucleic acid (mRNA) of MCP-1 was detected by either Northern blot or reverse-transcription polymerase chain reaction analysis in all cell lines and tumor specimens examined. In vivo expression of MCP-1 mRNA and protein was found predominantly in glioma cells with large and pleomorphic nuclei rather than in areas of small nucleated glioma cells. Adjacent brain tissue did not produce a significant level of MCP-1 mRNA or protein. Tumor vessels with endothelial proliferation expressed a moderate level of MCP-1 protein. Macrophages were found among the glioma cells, and the degree of macrophage infiltration was grossly correlated with the level of MCP-1 expression. The study results suggest that MCP-1 produced by the glioma cells may mediate macrophage infiltration into the glioma tissue.

Aged↗
PubMed1994

[Efficacy of the "Gen-Probe Mycobacterium Tuberculosis Direct Test (MTD)" for detection of Mycobacterium tuberculosis in clinical specimens--comparison between the MTD and the test by culture on Ogawa's egg medium or in the MB Check System].

Three or more weeks are usually required for detecting Mycobacterium tuberculosis by the known culture methods, and therefore, the development of a rapid bacteriological diagnostic method for M. tuberculosis has been urgently awaited. Recently, Gen-Probe Inc. has developed the "Gen-Probe Mycobacterium Tuberculosis Direct Test (MTD)", which is based on amplification of the ribosomal RNA (rRNA) of M. tuberculosis in clinical specimens and hybridization of the amplified rRNA with a M. tuberculosis-specific DNA probe, as a rapid direct diagnostic method for tuberculosis. We therefore compared the sensitivity and specificity of the MTD in detecting M. tuberculosis with the culture methods on Ogawa's egg medium and in the MB Check System, using 107 clinical specimens as test material. The results obtained are as follows: 1. Of the 61 clinical specimens which were negative when cultured on Ogawa's egg medium, 13 (21.3%) were positive for M. tuberculosis using the MTD, and of the 48 clinical specimens which were negative when cultured in the MB Check System, 8 specimens (16.7%) were positive for M. tuberculosis using the MTD. 2. All of the specimens which yielded growth of M. tuberculosis (identified by the DNA probe) either on Ogawa's egg medium or in the MB Check System, except one, were positive for M. tuberculosis in the MTD. The only one exception was a specimen which was positive for M. tuberculosis in both Ogawa's medium and the MB Check System.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗